Premium
Plasmid system for the intracellular production and purification of affinity‐tagged proteins in Bacillus megaterium
Author(s) -
Biedendieck Rebekka,
Yang Yang,
Deckwer WolfDieter,
Malten Marco,
Jahn Dieter
Publication year - 2006
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.21145
Subject(s) - bacillus megaterium , green fluorescent protein , fusion protein , tobacco etch virus , affinity chromatography , recombinant dna , biochemistry , biology , protease , isomerase , expression vector , bacillaceae , microbiology and biotechnology , chemistry , gene , enzyme , virus , bacteria , virology , plant virus , genetics , potyvirus , bacillus subtilis
Abstract A multiple vector system for the intracellular high‐level production of affinity tagged recombinant proteins in Bacillus megaterium was developed. The N‐ and C‐terminal fusion of a protein of interest to a Strep II and a His 6 ‐tag is possible. Corresponding genes are expressed under the control of a xylose‐inducible promoter in a xylose isomerase deficient host strain. The exemplatory protein production of green fluorescent protein (GFP) showed differences in produced and recovered protein amounts in dependence of the employed affinity tag and its N‐ or C‐terminal location. Up to 9 mg GFP per liter shake flask culture were purified using one‐step affinity chromatography. Integration of a protease cleavage site into the recombinant fusion protein allowed tag removal via tobacco etch virus (TEV) protease or Factor Xa treatment and a second affinity chromatographic step. Up to 274 mg/L culture were produced at 52 g CDW/L using a glucose limited fedbatch cultivation. GFP production and viability of the production host were followed by flow cytometry. Biotechnol. Bioeng. 2007;96: 525–537. © 2006 Wiley Periodicals, Inc.